Purine based fluorescent dyes

ABSTRACT

The present invention provides novel purine-based fluorescent dyes that may be used for staining, localizing and otherwise labeling target molecules, such as nucleic acids, for detection, amplification and quantification.

RELATED APPLICATIONS

This application is a continuation-in-part of U.S. patent applicationSer. No. 11/137,771, filed on May 24, 2005. The entirety of the contentsof Ser. No. 11/137,771 are hereby incorporated by reference.

FIELD OF THE INVENTION

This invention relates to a group of purine-based fluorescent dyes, andmore particularly, relates to fluorescent dyes which preferentiallystain nucleic acids.

All patents, patent applications, patent publications, scientificarticles and the like, cited or identified in this application arehereby incorporated by reference in their entirety in order to describemore fully the state of the art to which the present invention pertains.

BACKGROUND OF THE INVENTION

Fluorescent dyes that exhibit an enhanced fluorescence upon binding toDNA or RNA molecules are a basic tool in molecular and cell biology.Majority of the existing dyes bind noncovalently to nucleic acidsthrough a combination of hydrophobic interactions with the DNAbase-pairs and ionic binding to the negatively charged phosphatebackbone. The most useful dyes are preferably non-fluorescent in theabsence of the nucleic acids. Upon binding to DNA or RNA, however,several-fold fluorescence enhancement is measured, thereby enabling thedetection of small amounts of nucleic acids.

A variety of fluorescent dyes have been shown to be effective stains fornucleic acids. Examples of fluorescent DNA-binding dyes include ethidiumbromide, SYBR Green and SYBR Gold, which are commonly used to stain DNAin agarose gels, Hoechst 33258 and propidium iodide useful in flowcytometry or cell-impermeant TOTO dyes frequently used as a convenientindicator of apoptosis. Selected properties of these dyes such as thebrightness, spectral properties, resistance to photobleaching, affinityfor either single or double stranded nucleic acids can be modified tosome extend with changing the type and location of the substituents.

In order for a fluorescent dye to be a useful tool in molecular and cellbiology, the dye has to meet several requirements. First, it shouldpreferably have an excitation maximum between 300 and 900 nm, with aStoke shift of at least 10 nm. The most preferable dyes have anexcitation maximum between 500 and 900 nm. This spectroscopic range isboth compatible with the existing detection instrumentation and removedfrom any other interfering biological molecules. Fluorescent dyes usedin nucleic acid research should also have a high molar extinctioncoefficient, a high quantum yield, good photostability, andsignificantly increased fluorescence when bound to nucleic acids.

In U.S. Pat. No. 4,937,198, a purine-based fluorescent dye was disclosedthat selectively stained nucleic acids. The dye was reported to have amolar extinction coefficient of 60,000 M⁻¹ cm⁻¹, quantum yield of 0.4and excitation maximum in the presence of nucleic acids around 460 nmwith an emission maximum around 478 nm. Fluorescence enhancement of over7,000-fold was measured upon binding of the dye to RNA.

Moreda and Forrester (Tetrahedron, 53, 12595, 1997 and Tetrahedron, 53,12605, 1997) described a synthesis and characterization of a range ofpurine-based fluorescent dyes that were used as fluorescent dyes for DNAmarking in the diagnosis of malaria parasites.

SUMMARY OF THE INVENTION

The present application discloses novel purine-based dyes and theirmethods of preparation. In a preferred embodiment, the dyes are capableof selectively staining DNA and RNA, thereby providing high sensitivityfor detection of nucleic acids in solution or by gel analysis. Uponbinding of the dyes of the present invention to nucleic acids,fluorescence enhancement in excess of 1000-fold has been observed.Exemplary dyes showed a maximum absorbance upon binding to nucleic acidsaround 460-470 nm, and a broad fluorescence emission between 470 and 530nm. Some of the described dyes are characterized by the presence of apolycationic chain that may contribute towards a high binding affinitytowards nucleic acids, and others contain lipophylic moieties renderingthe dyes membrane permeable.

DESCRIPTION OF THE FIGURES

FIG. 1 illustrates a fluorescence enhancement of Dye 202 and Dye 203upon binding RNA. The experiment was carried out with concentration ofRNA 100 M/ml and 0.5 M Dye 202 or 203.

DESCRIPTION OF THE INVENTION

The present invention describes a group of novel, purine-basedfluorescent dyes that may be used as labels. In a preferred embodiment,the novel dyes are excitable around 460-470 nm, emit maximally around470-480 nm and selectively stain nucleic acids. In the absence ofnucleic acids, these particular dyes are essentially non-fluorescent.However, upon binding to nucleic acids, fluorescence enhancement inexcess of 1000-fold has been observed. The dyes are selective towardsnucleic acids, as only a very modest fluorescence enhancement isgenerated by the presence of proteins.

The dyes of the invention comprise three portions: a first portioncomprising a purinium ring system, a second portion comprising anaromatic ring system and a third portion which comprises a methinebridge joining the first and second portions. Examples of heterocyclicring systems that may be used for the second portion can include but notbe limited to benzazolium, pyridinium, quinolinium, lepidinium,acrydinium, pyrylium and thiopyrylium rings.

In one embodiment of the present invention, a purinium ring system isjoined to a benzazolium moiety and further comprises a charged or polargroup as described in U.S. patent application Ser. No. 11/137,771 (filedMay 24, 2005). As such these dyes have the structure:

wherein n can be 0, 1 or 2;

wherein Y comprises CR¹⁰R¹¹, NR¹⁰, O, S or Se where R¹⁰ and R¹¹independently comprise hydrogen, a halogen, an amino group, an alkylgroup wherein said alkyl group is saturated or unsaturated, linear orbranched, substituted or unsubstituted, an alkoxy group wherein saidalkyl group is saturated or unsaturated, branched or linear, substitutedor unsubstituted, or when taken together, R¹⁰ and R¹¹ form a 5 or 6membered ring;

wherein at least one of R¹, R², R³, R⁵, R⁶, R⁷, R⁸, R¹⁶, R¹⁷ or R¹⁸comprises Q; wherein Q comprises a sulfonate (SO₃ ⁻), a sulfonate ester(SO₂ER¹³), a sulfoxide (SOR¹³), a sulfone (SO₂CR¹³R¹⁴R¹⁵), a sulfonamide(SO₂NR¹³R¹⁴), a phosphate (PO₄ ⁼), a phosphate monoester (PO₃ ⁻ER¹³), aphosphate diester (PO₂ER¹³ER¹⁴), a phosphonate (PO₃ ⁼), a phosphonatemonoester (PO₂ ⁻ER¹³), a phosphonate diester (POER¹³ER¹⁴), athiophosphate (PSO₃ ⁼), a thiophosphate monoester (PSO₂ ⁻ER¹³), athiophosphate diester (PSOER¹³ER¹⁴), a thiophosphonate (PSO₂ ⁼), athiophosphonate monoester (PSO⁻ER¹³), a thiophosphonate diester(PSER¹³ER¹⁴), a phosphonamide (PONR¹³R¹⁴NR¹⁹R²⁰), its thioanalogue(PSNR¹³R¹⁴NR¹⁹R²⁰), a phosphoramide (PONR¹³R¹⁴NR¹⁵NR¹⁹R²⁰), itsthioanalogue (PSNR¹³R¹⁴NR¹⁵NR¹⁹R²⁰), a phosphoramidite (PO₂R¹⁹NR¹³R¹⁴)or its thioanalogue (POSR¹⁹NR¹³R¹⁴) wherein any of E can independentlycomprise O or S;

wherein Q is attached directly, or indirectly through a linker armcomprising carbon, sulfur, oxygen, nitrogen, and any combinationsthereof and wherein said linker arm may be saturated or unsaturated,linear or branched, substituted or unsubstituted and any combinationsthereof;

wherein the remaining R¹, R², R³, R⁵, R⁶, R⁷, R⁸, R¹⁶, R¹⁷ and R¹⁸independently comprise hydrogen, Z, an alkyl group wherein said alkylgroup is saturated or unsaturated, linear or branched, substituted orunsubstituted, an alkoxy group wherein said alkoxy group is saturated orunsaturated, branched or linear, substituted or unsubstituted, or whentaken together, R¹⁶ and R³, R³ and R⁸, R⁸ and R⁷, R⁷ and R⁶, R⁶ and R⁵,R⁵ and R¹⁰, R¹⁶ and R¹⁷, R¹⁰ and R¹⁶, and R¹⁷ and R¹⁸ may form a 5 or 6membered ring;

wherein Z comprises a carboxyl group (CO₂ ⁻), a carbonate ester(COER¹³), a sulfonate (SO₃ ⁻), a sulfonate ester (SO₂ER¹³), a sulfoxide(SOR¹³), a sulfone (SO₂CR¹³R¹⁴R¹⁵), a sulfonamide (SO₂NR¹³R¹⁴), aphosphate (PO₄ ⁼), a phosphate monoester (PO₃ ⁻ER¹³), a phosphatediester (PO₂ER¹³ER¹⁴), a phosphonate (PO₃ ⁼), a phosphonate monoester(PO₂ ⁻ER¹³), a phosphonate diester (POER¹³ER¹⁴), a thiophosphate (PSO₃⁼), a thiophosphate monoester (PSO₂ ⁻ER¹³), a thiophosphate diester(PSOER¹³ER¹⁴), a thiophosphonate (PSO₂ ⁼), a thiophosphonate monoester(PSO⁻ER¹³), a thiophosphonate diester (PSER¹³ER¹⁴), a phosphonamide(PONR¹³R¹⁴NR¹⁹R²⁰), its thioanalogue (PSNR¹³R¹⁴NR¹⁹R²⁰), a phosphoramide(PONR¹³R¹⁴NR¹⁵NR¹⁹R²⁰), its thioanalogue (PSNR¹³R¹⁴NR¹⁵NR¹⁹R²⁰), aphosphoramidite (PO₂R¹⁹NR¹³R¹⁴) or its thioanalogue (POSR¹⁹NR¹³R¹⁴)where E can be independently O or S;

wherein Z is attached directly, or indirectly through a linker armcomprising carbon, sulfur, oxygen, nitrogen, and any combinationsthereof and wherein said linker arm may be saturated or unsaturated,linear or branched, substituted or unsubstituted and any combinationsthereof;

and wherein any of R¹, R², R³, R⁵, R⁶, R⁷, R⁸, R¹⁶, R¹⁷ and R¹⁸ mayindependently further comprise a heteroatom containing side chainwherein said side chain is joined to the R group by a linkage whichcomprises an ether linkage (—OR²⁵), a thioether linkage (—SR²⁵), or anamine linkage (—NR²⁵R²⁶ or —N⁺R²⁵R²⁶R²⁷), and wherein R²⁵, R²⁶ and R²⁷independently comprise hydrogen, Z, an alkyl group wherein said alkylgroup is saturated or unsaturated, linear or branched, substituted orunsubstituted, an alkoxy group that is saturated or unsaturated,branched or linear, substituted or unsubstituted, or when takentogether, R²⁵ and R²⁶, and R²⁶ and R²⁷ independently comprise a five orsix membered ring, and wherein any of R²⁵, R²⁶ or R²⁷ may furthercomprise said heteroatom containing side chain.

In prior art, benzazolium moieties have been joined to purinium rings(U.S. Pat. No. 4,937,198, and Moreda and Forrester supra) but withoutthe advantages endowed by the presence of the charged or polar groupsdescribed as Q above.

In another embodiment of the present invention, the second portionjoined to the purinium first portion results in dyes with the structure:

wherein n is 0, 1 or 2;

wherein Y is —CR⁶═CR⁷—;

wherein m and p have values of 0 or 1 and m+p=1;

wherein R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R¹⁶, R¹⁷ and R¹⁸ independentlycomprise hydrogen, Z, an alkyl group wherein said alkyl group issaturated or unsaturated, linear or branched, substituted orunsubstituted, an alkoxy group wherein said alkoxy group is saturated orunsaturated, branched or linear, substituted or unsubstituted, or whentaken together, a combination or combinations of R¹, R², R³, R⁴, R⁵, R⁶,R⁷, R¹⁶, R¹⁷ and R¹⁸ form one or more 5 or 6 membered rings;

wherein Z comprises a carboxyl group (CO₂ ⁻), a carbonate ester(COER¹³), a sulfonate (SO₃ ⁻), a sulfonate ester (SO₂ER¹³), a sulfoxide(SOR¹³), a sulfone (SO₂CR¹³R¹⁴R¹⁵), a sulfonamide (SO₂NR¹³R¹⁴), aphosphate (PO₄ ⁼), a phosphate monoester (PO₃ ⁻ER¹³), a phosphatediester (PO₂ER¹³ER¹⁴), a phosphonate (PO₃ ⁼), a phosphonate monoester(PO₂ ⁻ER¹³), a phosphonate diester (POER¹³ER¹⁴), a thiophosphate (PSO₃⁼), a thiophosphate monoester (PSO₂ ⁻ER¹³), a thiophosphate diester(PSOER¹³ER¹⁴), a thiophosphonate (PSO₂ ⁼), a thiophosphonate monoester(PSO⁻ER¹³), a thiophosphonate diester (PSER¹³ER¹⁴), a phosphonamide(PONR¹³R¹⁴NR¹⁹R²⁰), its thioanalogue (PSNR¹³R¹⁴NR¹⁹R²⁰), a phosphoramide(PONR¹³R¹⁴NR¹⁵NR¹⁹R²⁰), its thioanalogue (PSNR¹³R¹⁴NR¹⁵NR¹⁹R²⁰), aphosphoramidite (PO₂R¹⁹NR¹³R¹⁴) or its thioanalogue (POSR¹⁹NR¹³R¹⁴)where E can be independently O or S;

wherein Z is attached directly, or indirectly through a linker armcomprising carbon, sulfur, oxygen, nitrogen, and any combinationsthereof and wherein said linker arm may be saturated or unsaturated,linear or branched, substituted or unsubstituted and any combinationsthereof;

and wherein any of R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R¹⁶, R¹⁷ and R¹⁸ mayindependently further comprise a heteroatom containing side chainwherein said side chain is joined to the R group by a linkage whichcomprises an ether linkage (—OR²⁵), a thioether linkage (—SR²⁵), or anamine linkage (—NR²⁵R²⁶ or —N⁺R²⁵R²⁶R²⁷), and wherein R²⁵, R²⁶ and R²⁷independently comprise hydrogen, Z, an alkyl group wherein said alkylgroup is saturated or unsaturated, linear or branched, substituted orunsubstituted, an alkoxy group that is saturated or unsaturated,branched or linear, substituted or unsubstituted, or when takentogether, R²⁵ and R²⁶, and R²⁶ and R²⁷ independently comprise a five orsix membered ring, and wherein any of R²⁵, R²⁶ or R²⁷ may furthercomprise said heteroatom containing side chain.

In this aspect of the present invention, the dyes described above mayenjoy the benefits of being modified by groups described as Z or theymay be used without such groups.

In another embodiment of the present invention, a different aromaticgroup is chosen as the second portion resulting in a composition withthe structure:

wherein n is 0, 1 or 2;

wherein Y is either S or O;

wherein R¹, R², R³, R⁴, R⁵, R⁶, R¹⁶, R¹⁷ and R¹⁸ independently comprisehydrogen, Z, an alkyl group wherein said alkyl group is saturated orunsaturated, linear or branched, substituted or unsubstituted, an alkoxygroup wherein said alkoxy group is saturated or unsaturated, branched orlinear, substituted or unsubstituted, or when taken together, acombination or combinations of R¹, R², R³, R⁴, R⁵, R⁶, R¹⁶, R¹⁷ and R¹⁸form one or more 5 or 6 membered rings;

wherein Z comprises a carboxyl group (CO₂ ⁻), a carbonate ester(COER¹³), a sulfonate (SO₃ ⁻), a sulfonate ester (SO₂ER¹³), a sulfoxide(SOR¹³), a sulfone (SO₂CR¹³R¹⁴R¹⁵), a sulfonamide (SO₂NR¹³R¹⁴), aphosphate (PO₄ ⁼), a phosphate monoester (PO₃ ⁻ER¹³), a phosphatediester (PO₂ER¹³ER¹⁴), a phosphonate (PO₃ ⁼), a phosphonate monoester(PO₂ ⁻ER¹³), a phosphonate diester (POER¹³ER¹⁴), a thiophosphate (PSO₃⁼), a thiophosphate monoester (PSO₂ ⁻ER¹³), a thiophosphate diester(PSOER¹³ER¹⁴), a thiophosphonate (PSO₂ ⁼), a thiophosphonate monoester(PSO⁻ER¹³), a thiophosphonate diester (PSER¹³ER¹⁴), a phosphonamide(PONR¹³R¹⁴NR¹⁹R²⁰), its thioanalogue (PSNR¹³R¹⁴NR¹⁹R²⁰), a phosphoramide(PONR¹³R¹⁴NR¹⁵NR¹⁹R²⁰), its thioanalogue (PSNR¹³R¹⁴NR¹⁵NR¹⁹R²⁰), aphosphoramidite (PO₂R¹⁹NR¹³R¹⁴) or its thioanalogue (POSR¹⁹NR¹³R¹⁴)where E can be independently O or S;

wherein Z is attached directly, or indirectly through a linker armcomprising carbon, sulfur, oxygen, nitrogen, and any combinationsthereof and wherein said linker arm may be saturated or unsaturated,linear or branched, substituted or unsubstituted and any combinationsthereof;

and wherein any of R¹, R², R³, R⁴, R⁵, R⁶, R¹⁶, R¹⁷ and R¹⁸ mayindependently further comprise a heteroatom containing side chainwherein said side chain is joined to the R group by a linkage whichcomprises an ether linkage (—OR²⁵), a thioether linkage (—SR²⁵), or anamine linkage (—NR²⁵R²⁶ or —N⁺R²⁵R²⁶R²⁷), and wherein R²⁵, R²⁶ and R²⁷independently comprise hydrogen, Z, an alkyl group wherein said alkylgroup is saturated or unsaturated, linear or branched, substituted orunsubstituted, an alkoxy group that is saturated or unsaturated,branched or linear, substituted or unsubstituted, or when takentogether, R²⁵ and R²⁶, and R²⁶ and R²⁷ independently comprise a five orsix membered ring, and wherein any of R²⁵, R²⁶ or R²⁷ may furthercomprise said heteroatom containing side chain.

In this aspect of the present invention, the dyes described above mayenjoy the benefits of being modified by groups described as Z or theymay be used without such groups.

In another embodiment of the present invention, a benzazole group ischosen as the second portion and the methine group is modified bybridging a portion of it to the benzazole moiety resulting in acomposition with the structure:

wherein m is 1, 2, 3, or 4;

wherein n is 0, 1 or 2;

wherein Y comprises CR¹⁰OR¹¹, NR¹⁰, O, S or Se where R¹⁰ and R¹¹independently comprise hydrogen, a halogen, an amino group, an alkylgroup wherein said alkyl group is saturated or unsaturated, linear orbranched, substituted or unsubstituted, an alkoxy group wherein saidalkyl group is saturated or unsaturated, branched or linear, substitutedor unsubstituted, or when taken together, R¹⁰ and R¹¹ form a 5 or 6membered ring;

wherein R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R¹⁶ and R¹⁷ independentlycomprise hydrogen, Z, an alkyl group wherein said alkyl group issaturated or unsaturated, linear or branched, substituted orunsubstituted, an alkoxy group wherein said alkoxy group is saturated orunsaturated, branched or linear, substituted or unsubstituted, or whentaken together, a combination or combinations of R¹, R², R³, R⁴, R⁵, R⁶,R⁷, R⁸, R¹⁶, and R¹⁷ form one or more 5 or 6 membered rings;

wherein Z comprises a carboxyl group (CO₂ ⁻), a carbonate ester(COER¹³), a sulfonate (SO₃ ⁻), a sulfonate ester (SO₂ER¹³), a sulfoxide(SOR¹³), a sulfone (SO₂CR¹³R¹⁴R¹⁵), a sulfonamide (SO₂NR¹³R¹⁴), aphosphate (PO₄ ⁼), a phosphate monoester (PO₃ ⁻ER¹³), a phosphatediester (PO₂ER¹³ER¹⁴), a phosphonate (PO₃ ⁼), a phosphonate monoester(PO₂ ⁻ER¹³), a phosphonate diester (POER¹³ER¹⁴), a thiophosphate (PSO₃⁼), a thiophosphate monoester (PSO₂ ⁻ER¹³), a thiophosphate diester(PSOER¹³ER¹⁴), a thiophosphonate (PSO₂ ⁼), a thiophosphonate monoester(PSO⁻ER¹³), a thiophosphonate diester (PSER¹³ER¹⁴), a phosphonamide(PONR¹³R¹⁴NR¹⁹R²⁰), its thioanalogue (PSNR¹³R¹⁴NR¹⁹R²⁰), a phosphoramide(PONR¹³R¹⁴NR¹⁵NR¹⁹R²⁰), its thioanalogue (PSNR¹³R¹⁴NR¹⁵NR¹⁹R²⁰), aphosphoramidite (PO₂R¹⁹NR¹³R¹⁴) or its thioanalogue (POSR¹⁹NR¹³R¹⁴)where E can be independently O or S;

wherein Z is attached directly, or indirectly through a linker armcomprising carbon, sulfur, oxygen, nitrogen, and any combinationsthereof and wherein said linker arm may be saturated or unsaturated,linear or branched, substituted or unsubstituted and any combinationsthereof;

and wherein any of R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R¹⁶, and R¹⁷ mayindependently further comprise a heteroatom containing side chainwherein said side chain is joined to the R group by a linkage whichcomprises an ether linkage (—OR²⁵), a thioether linkage (—SR²⁵), or anamine linkage (—NR²⁵R²⁶ or —N⁺R²⁵R²⁶R²⁷), and wherein R²⁵, R²⁶ and R²⁷independently comprise hydrogen, Z, an alkyl group wherein said alkylgroup is saturated or unsaturated, linear or branched, substituted orunsubstituted, an alkoxy group that is saturated or unsaturated,branched or linear, substituted or unsubstituted, or when takentogether, R²⁵ and R²⁶, and R²⁶ and R²⁷ independently comprise a five orsix membered ring, and wherein any of R²⁵, R²⁶ or R²⁷ may furthercomprise said heteroatom containing side chain.

In this aspect of the present invention, the dyes described above mayenjoy the benefits of being modified by groups described as Z or theymay be used without such groups. Rigidization of the methine bond hasbeen previously described in joining together two benzazolium moieties(U.S. Pat. Nos. 6,133,445 and 6,686,145 both of which are herebyincorporated by reference) but has not been previously described injoining a benzazolium to a purinium dye.

The novel dyes of the present invention may be used for any purposespreviously described for fluorescent dyes. For example, these dyes maybe modified with a reactive group to attach them to target molecules ofinterest. These dyes may also be used free in solution and advantagetaken of their ability to bind to target molecules of interest. Ofespecial use is when this binding event results in an increase influorescence of the dye molecules. For further examples of use,reference is made to U.S. patent application Ser. No. 11/137,771 filedMay 24, 2005, hereby incorporated by reference.

The examples which follow are set forth to illustrate various aspects ofthe present invention but are not intended in any way to limit its scopeas more particularly set forth and defined in the claims that followthereafter.

DESCRIPTION OF THE PREFERRED EMBODIMENTS EXAMPLE 1 Synthesis of Dye 201(a) Preparation of 4-(Sulfobutyl)-2-methylbenzothiazole (Compound 1)

A mixture of 2-methylbenzothiazole (1.49 g, 10 mmol) and 1,4-butanesultone (4.0 g, 30 mmol) was heated in a pressure tube at 130-140° C.for 3 hours. The mixture was allowed to cool to room temperature, andthe resulting mass was triturated with ethyl acetate (50 ml) until agray solid separated. The solid was collected by centrifugation, washedwith ethyl acetate and dried under vacuum to yield 2.8 g (96%) ofCompound 1.

(b) Preparation of 3-Methyl-6-(methylthio)purine (Compound 2)

Compound 2 was prepared as described in U.S. Pat. No. 4,937,198 (hereinincorporated by reference). The structure of this compound is givenabove.

(c) Preparation of 7-(Iodopropyl)-3-methyl-6-(methylthio)purinium iodide(Compound 3)

A pressure tube, equipped with a magnetic stirring bar, was charged withCompound 2 (0.54 g, 3 mmole) and 1,3-diiodopropane (2.66 g, 9 mmole).The mixture was heated to ˜120° C. for 3 hours. The residue wasextensively washed with ethyl acetate and diethyl ether and dried undervacuum to give 0.4 g of yellow solid Compound 3 (yield ˜28%).

(d) Preparation of Dye 201

To a pear-shaped flask, equipped with a magnetic stirring bar and areflux condenser, were added Compound 1 (72 mg, 250 mole), Compound 3(120 mg, 250 mole), 2 ml of methanol and 42 l of triethylamine. Themixture was refluxed for about 45 minutes, producing a red solutioncontaining an orange solid. The contents of the flask were cooled andthe reaction mixture was added to 50 ml ethyl acetate. The resultingyellow precipitate was centrifuged and extensively washed with ethylacetate. The residue was dried under vacuum to give the title compound(55% yield).

EXAMPLE 2 Synthesis of Dye 202

A mixture of Dye 201 obtained in Example 1 (50 mg, 70 mole),N,N,N′,N′-tetramethyldiaminobutane (6.5 l, 35 mole) and 3 ml of ethanolwas refluxed for 2 hours, producing a red solution. The contents of theflask were cooled and the reaction mixture was added to 50 ml ethylacetate. The resulting red precipitate was centrifuged and extensivelywashed with ethyl acetate followed by washing with diethyl ether. Theresidue was dried under vacuum to give Dye 202 (48% yield).

EXAMPLE 3 Synthesis of Dye 203

A pear-shaped flask, equipped with a magnetic stirring bar and a refluxcondenser, was charged with—Compound 1 from step (a) of Example 1 (90mg, 315 mole), Compound 3 from step (c) of Example 1 (150 mg, 315 mole),N,N,N′,N′-tetramethyldiaminobutane (0.58 ml, 3.15 mmole) and 2.5 ml ofmethanol. The mixture was refluxed for about 2 hours, producing abrownish solution containing an orange solid. The contents of the flaskwere cooled and the reaction mixture was added to 50 ml ethyl acetate.The resulting brown precipitate was centrifuged and extensively washedwith ethyl acetate. The residue was dried under vacuum to give Dye 203(67% yield).

EXAMPLE 4 Synthesis of Dye 204 (a) Preparation ofN-(2,3-dimethylbenzothiazole-6-sulfonyl)piperidine tosylate (compound 4)

Compound 4 was prepared as described in U.S. patent application Ser. No.11/137,771 filed May 24, 2005 (herein incorporated by reference). Thestructure of this compound is given above.

(b) Preparation of 3,7-Dimethyl-6-(methylthio)purinium tosylate(Compound 5)

Compound 5 was prepared as described in U.S. Pat. No. 4,937,198 (hereinincorporated by reference). The structure of this compound is givenabove.

(c) Preparation of Dye 204

A mixture of Compound 4 (64 mg, 136 mole), Compound 5 (50 mg, 136 mole)and 20 l of triethylamine was refluxed for about 1 hour, producing anorange solution. The contents of the flask were cooled and the reactionmixture was added to 50 ml ethyl acetate. The resulting orangeprecipitate was centrifuged and extensively washed with ethyl acetate.The residue was dried under vacuum to give Dye 204 (61% yield).

EXAMPLE 5 Interaction of Dyes 202 and 203 with Nucleic Acids

a) Absorbance Measurements

Dyes 202 and 203 were dissolved in DMSO to give 609 M and 1.35 mMsolutions, respectively. These stock solutions were diluted to a dyeconcentration of 20 M either with water alone or with water containingRNA (S. cerevisiae, Sigma-Aldrich) at a concentration of 1 mg/ml andabsorbance readings were taken. The absorbance maximum in the absence ofRNA was at 452 nm, while the maximum in the presence of RNA was shiftedto 462 nm.

b) Emission Measurements

The above mentioned stocks were diluted with DMSO to prepare 100 Msolutions of Dyes 202 and 203. Subsequently, 5 l of dye solution wasmixed with either 995 l of RNase-free water or 995 l water containing100 l RNA (1 mg/ml). The solutions were incubated for 5 minutes at roomtemperature and the fluorescence emission was measured with anexcitation wavelength of 463 nm. As seen in FIG. 1, virtually nofluorescence was observed in the absence of RNA while a broad emissioncurve was measured for RNA-dye complexes.

EXAMPLE 6 Interaction of Dyes 202 and 203 with Proteins

Dyes 202 and 203 stock solutions from Example 5 were diluted with waterto give 100 M solutions of Dyes 202 and 203. Subsequently, 5 l of the100 M dye solution was incubated in 10 mM TrisXHCl buffer (pH 7.2) inthe absence or in the presence of 1 ml of 100 g/ml of BSA, lysozyme orcasein. The fluorescence emission was measured with an excitationwavelength of 463 nm. Virtually no fluorescence was observed in theabsence of proteins. When the dyes were incubated in the presence ofproteins only a modest increase in fluorescence intensity was recorded(data not shown) indicating essentially a lack of signal by the presenceof proteins and a specificity of signal enhancement by nucleic acids forthese dyes.

FIG. 1

Many obvious variations will be suggested to those of ordinary skill inthe art in light of the above detailed descriptions of the presentinvention. All such obvious variations are fully contemplated and areembraced by the scope and spirit of the present invention as set forthin the claims that now follow.

1. A dye having the formula:

wherein n is 0, 1 or 2; wherein Y comprises CR¹⁰R¹¹, NR¹⁰, O, S or Sewhere R¹⁰ and R¹¹ independently comprise hydrogen, a halogen, an aminogroup, an alkyl group wherein said alkyl group is saturated orunsaturated, linear or branched, substituted or unsubstituted, an alkoxygroup wherein said alkoxy group is saturated or unsaturated, branched orlinear, substituted or unsubstituted, or when taken together, R¹⁰ andR¹¹ form a 5 or 6 membered ring; wherein at least one of R¹, R², R³, R⁵,R⁶, R⁷, R⁸, R¹⁶, R¹⁷ or R¹⁸ comprises Q; wherein Q comprises a sulfonateester (SO₂ER¹³), a sulfoxide (SOR¹³), a sulfone (SO₂CR¹³R¹⁴R¹⁵), asulfonamide (SO₂NR¹³R¹⁴), a phosphate (PO₄ ⁼), a phosphate monoester(PO₃ ⁻ER¹³), a phosphate diester (PO₂ER¹³ER¹⁴), a phosphonate (PO₃ ⁼), aphosphonate monoester (PO₂ ⁻ER^(—)), a phosphonate diester (POER¹³ER¹⁴),a thiophosphate (PSO₃ ⁼), a thiophosphate monoester (PSO₂ ⁻ER¹³), athiophosphate diester (PSOER¹³ER¹⁴), a thiophosphonate (PSO₂ ⁼), athiophosphonate monoester (PSO⁻ER¹³), a thiophosphonate diester(PSER¹³ER¹⁴), a phosphonamide (PONR¹³R¹⁴NR¹⁹R²⁰), its thioanalogue(PSNR¹³R¹⁴NR¹⁹R²⁰), a phosphoramide (PONR¹³R¹⁴NR¹⁵NR¹⁹R²⁰), itsthioanalogue (PSNR¹³R¹⁴NR¹⁵NR¹⁹R²⁰), a thioanalogue of phosphoramidite(POSR¹⁹NR¹³R¹⁴), wherein any of E can independently comprise O or S;wherein Q is attached directly, or indirectly through a linker armcomprising carbon, sulfur, oxygen, nitrogen, or any combinationsthereof, and wherein said linker arm may be saturated or unsaturated,linear or branched, substituted or unsubstituted, or any combinationsthereof; wherein the remaining R¹, R², R³, R⁵, R⁶, R⁷, R⁸, R¹⁶, R¹⁷ andR¹⁸ independently comprise hydrogen, Z, an alkyl group wherein saidalkyl group is saturated or unsaturated, linear or branched, substitutedor unsubstituted, an alkoxy group wherein said alkoxy group is saturatedor unsaturated, branched or linear, substituted or unsubstituted, orwhen taken together, R¹⁶ and R³, R³ and R⁸, R⁸ and R⁷, R⁷ and R⁶, R⁶ andR⁵, R⁵ and R¹⁰, R¹⁶ and R¹⁷, R¹⁰ and R¹⁶, and R¹⁷ and R¹⁸ may form a 5or 6 membered ring; wherein Z comprises a carboxyl group (CO₂ ⁻), acarbonate ester (COER¹³), a sulfonate (SO₃ ⁻), a sulfonate ester(SO₂ER¹³), a sulfoxide (SOR¹³), a sulfone (SO₂CR¹³R¹⁴R¹⁵), a sulfonamide(SO₂NR¹³R¹⁴), a phosphate (PO₄ ⁼), a phosphate monoester (PO₃ ⁻ER¹³), aphosphate diester (PO₂ER¹³ER¹⁴), a phosphonate (PO₃ ⁼), a phosphonatemonoester (PO₂ ⁻ER¹³), a phosphonate diester (PO₂ER¹³ER¹⁴), athiophosphate (PSO₃ ⁼), a thiophosphate monoester (PSO₂ ⁻ER¹³), athiophosphate diester (PSOER¹³ER¹⁴), a thiophosphonate (PSO₂ ⁼), athiophosphonate monoester (PSO⁻ER¹³), a thiophosphonate diester(PSER¹³ER¹⁴), a phosphonamide (PONR¹³R¹⁴NR¹⁹R²⁰), its thioanalogue(PSNR¹³R¹⁴NR¹⁹R²⁰), a phosphoramide (PONR¹³R¹⁴NR¹⁵NR¹⁹R²⁰), itsthioanalogue (PSNR¹³R¹⁴NR¹⁵NR¹⁹R²⁰), a phosphoramidite (PO₂R¹⁹NR¹³R¹⁴)or its thioanalogue (POSR¹⁹NR¹³R¹⁴), wherein any of E can independentlycomprise O or S; wherein Z is attached directly, or indirectly through alinker arm comprising carbon, sulfur, oxygen, nitrogen, or anycombinations thereof, and wherein said linker arm may be saturated orunsaturated, linear or branched, substituted or unsubstituted, or anycombinations thereof; and wherein any of R¹, R², R³, R⁵, R⁶, R⁷, R⁸,R¹⁶, R¹⁷ and R¹⁸ may independently further comprise a heteroatomcontaining side chain wherein said side chain is joined to said R groupby a linkage which comprises an ether linkage (—OR²⁵), a thioetherlinkage (—SR²⁵), or an amine linkage (—NR²⁵R²⁶ or —N⁺R²⁵R²⁶R²⁷), andwherein R²⁵, R²⁶ and R²⁷ independently comprise hydrogen, Z, an alkylgroup wherein said alkyl group is saturated or unsaturated, linear orbranched, substituted or unsubstituted, an alkoxy group that issaturated or unsaturated, branched or linear, substituted orunsubstituted, or when taken together, R²⁵ and R²⁶, and R²⁶ and R²⁷independently comprise a five or six membered ring, and wherein any ofR²⁵, R²⁶ or R²⁷ may further comprise said heteroatom containing sidechain.
 2. The dye of claim 1, wherein at least one of R¹, R², R³, R⁵,R⁶, R⁷, R⁸, R¹⁰, R¹¹, R¹³, R¹⁴, R¹⁵, R¹⁶, R¹⁷ or R¹⁸ comprises areactive group.
 3. The dye of claim 2, wherein said reactive groupcomprises a nucleophilic reactive group, an electrophilic reactivegroup, a terminal alkene, a terminal alkyne, a coordinate group or analkylating agent.
 4. The dye of claim 3, wherein said nucleophilicreactive group comprises a thiol, amine, hydrazine or hydroxyl group. 5.The dye of claim 3, wherein said electrophilic reactive group comprisesan isocyanate, isothiocyanate, monochlorotriazine, dichlorotriazine,4,6-dichloro-1,3,5-triazines, mono- or di-halogen substituted pyridine,mono- or di-halogen substituted diazine, maleimide, haloacetamide,aziridine, sulfonyl halide, acid halide, hydroxysuccinimide ester,hydroxysulfosuccinimide ester, imido ester, azidonitrophenol, azide,3-(2-pyridyl dithio)proprionamide, glyoxal or aldehyde group.
 6. The dyeof claim 1, wherein said dye is linked to a target molecule.
 7. The dyeof claim 6, wherein said target molecule comprises a nucleoside,nucleotide, oligonucleotide, polynucleotide, peptide nucleic acid,protein, peptide, enzyme, antigen, antibody, hormone, hormone receptor,cellular receptor, lymphokine, cytokine, hapten, lectin, avidin,strepavidin, digoxygenin, carbohydrate, oligosaccharide, polysaccharide,lipid, glycolipid, viral particle, viral component, bacterial cell,bacterial component, eucaryotic cell, eukaryotic cell component, naturaldrug, synthetic drug, glass particle, glass surface, plastic particle,plastic surface, siliceous particle, siliceous surface, organicmolecule, dye or a derivative thereof.
 8. The dye of claim 7, whereinsaid nucleoside, nucleotide, oligonucleotide, or polynucleotidecomprises one or more ribonucleoside moieties, ribonucleotide moieties,deoxyribonucleoside moieties, deoxyribonucleotide moieties, modifiedribonucleosides, modified ribonucleotides, modifieddeoxyribonucleosides, modified deoxyribonucleotides, ribonucleotideanalogues, deoxyribonucleotide analogues or any combination thereof. 9.The dye of claim 6, wherein said dye is linked to said target moleculethrough a linker arm.
 10. The dye of claim 9, wherein said linker arm isattached to said target molecule through a bond which comprises acovalent bond, a non-covalent bond, a polar bond or a coordinate bond.11. The oligonucleotide or polynucleotide of claim 8, wherein saidoligonucleotide or polynucleotide comprises two or more dye moleculesattached to separate nucleotides of said oligonucleotide orpolynucleotide.
 12. The oligonucleotide or polynucleotide of claim 11,wherein said two or more dye molecules comprise the same dye molecules.13. The oligonucleotide or polynucleotide of claim 11, wherein said twoor more dye molecules comprise different dye molecules.
 14. A compositedye comprising the dye of claim 7, said dye being joined or attached toa second dye molecule.
 15. The dye of claim 1, wherein said dye isattached to a target specific moiety.
 16. The dye of claim 15, whereinsaid target specific moiety comprises a protein or a nucleic acid. 17.The dye of claim 16, wherein said protein comprises an antibody or afragment thereof.
 18. The dye of claim 16, wherein said nucleic acidcomprises unmodified nucleotides, modified nucleotides, nucleotideanalogues or any combination thereof.
 19. The dye of claim 1, whereinsaid alkyl or alkoxy groups independently comprise from 1-18 carbons.20. The dye of claim 19, wherein said alkyl or alkoxy groupsindependently comprise from 1-6 carbons.
 21. The dye of claim 3 whereinsaid electrophilic reactive group comprises an isocyanate,isothiocyanate, monochlorotriazine, dichlorotriazine,4,6-dichloro-1,3,5-triazines, mono- or di-halogen substituted pyridine,mono- or di-halogen substituted diazine, maleimide, haloacetamide,aziridine, sulfonyl halide, acid halide, hydroxysuccinimide ester,hydroxysulfosuccinimide ester, imido ester, azidonitrophenol, azide,3-(2-pyridyl dithio)proprionamide, glyoxal or aldehyde group.
 22. A dyehaving the formula

or


23. A dye having the formula